PROJECT SUMMARY The human genome is constantly exposed to a variety of genotoxic agents that result in thousands of DNA lesions in each cell every day. To cope with these lesions, cells activate a series of signaling cascades that are collectively known as the DNA damage response (DDR). DDR pathways function to recognize and repair DNA lesions, but also to coordinate DNA repair with cell cycle progression. Defects in these pathways result in genomic instability, a hallmark of cancer, and also cause other diseases such as neurological and developmental disorders. The ATR checkpoint kinase is a master regulator of the DDR, and in response to DNA damage, phosphorylates hundreds of downstream targets. Activation of ATR signaling is paramount for maintaining genome stability, as it allows cells to precisely coordinate the processes of DNA replication and DNA repair, and to ensure that DNA replication has completed prior to mitotic entry. ATR activation requires the assembly of a multi-protein complex that includes at least one ATR activating protein. In the budding yeast Saccharomyces cerevisiae, there are three activators of the ATR orthologue, Mec1. Extensive research on the Mec1 activators has revealed that these proteins all activate Mec1 using a similar mechanism, but that they function at least somewhat non-redundantly in cells. Mammalian cells possess at least two ATR activators, TOPBP1 and ETAA1. Similar to the Mec1 activators, current evidence suggests that TOPBP1 and ETAA1 activate ATR in the same manner but have non-redundant cellular functions. The overall goals of this project are to test this hypothesis further and better understand why cells have two ATR activators. Aim 1 is to determine the molecular mechanism by which TOPBP1 and ETAA1 activate ATR, and Aim 2 is to identify differences between TOPBP1- and ETAA1-dependent ATR signaling in cells. ATR signaling has recently emerged as a promising therapeutic target in cancer with the development of small molecule ATR inhibitors. Further delineating mechanisms of ATR activation and outcomes of ATR signaling will help to maximize the efficacy of these drugs and aide in the identification of patients who would most likely benefit from ATR inhibitor treatment regimens.